The yeast Saccharomyces cerevisiae has proven to be a model eukaryotic organism for many types of studies, including gene regulation. Importantly, the transcription regulatory machinery is conserved between yeast vertebrates, and insights gained from studies in yeast are generally universal. Studies of the yeast HO genes have identified many important transcriptional regulators and helped characterize regulatory paradigms. Chromatin immunoprecipitation (CHIP) assays have recently shown that activation of HO proceeds in a series of steps, with the Swi5 DNA-binding factor required for subsequent recruitment of the Swi/SNF and SAGA chromatin modulating complexes and the SBF factor. The investigator has identified several additional factors required for activation of HO, and our experiments suggest an important role for a DNA loop in transcriptional activation. CHIP experiments are proposed to examine the mechanisms of HO regulation. Specific regions of the Swi5 protein are implicated in recruiting Swi/Snf and SAGA to the HO promoter and both genetic and biochemical experiments are proposed to examine this further. Sin4 is present in two transcriptional regulatory complexes, the PolII mediator and the SAGA acetyltransferase. A sin4 null mutation causes pleiotropic effects on transcription and chromatin, with expression of some genes increased and others decreased. He will conduct a genetic screen to obtain two classes of sin4 mutant alleles, mutations that are defective for only SAGA function or only mediator. These specific alleles will be characterized genetically and used in suppressor screens. DNA microarray experiments will provide global information about how mutations in these two transcriptional regulatory complexes affect patterns of gene expression. Immunoprecipitation experiments will provide information on protein interactions in these complexes, and biochemical experiments will determine how these mutant alleles affect histone acetylation and transcription in vitro. The Sin3 transcriptional repressor is in a protein complex with the Rpd3 histone deacetylase (HDAC). Sin3 can recognize DNA-binding proteins through one of four PAH motifs, and thus targets the repression complex to specific promoters. Strains have been constructed with mutations in these PAH motifs and DNA microarray experiments will allow us to determine which PAH protein interaction motif is required for repression of which gene. These results can be combined with a screen to identify DNA-binding proteins that repress transcription in a SIN3-dependent manner. Preliminary studies have identified several mutations that may affect repression by Sin3 or interactions between Sin3 and Rpd3, and these will be characterized further. Transcriptional repression by Sin3 requires more than just recruiting an HDAC, and our data suggest that the SAGA histone acetyltransferase is required for full repression by Sin3. Experiments with mutant histones are proposed to examine the role of histone acetylation in Sin3 repression. Genetic screens will identify components of HDAC-independent repression by Sin3 and proteins that may regulate Sin3 repressive activity.